Natriuretic Peptide Receptor A as a Novel Target for Prostate Cancer

Department of Molecular Medicine, University of South Florida, Tampa, FL 33612, USA.
Molecular Cancer (Impact Factor: 4.26). 05/2011; 10(1):56. DOI: 10.1186/1476-4598-10-56
Source: PubMed
ABSTRACT
The receptor for the cardiac hormone atrial natriuretic peptide (ANP), natriuretic peptide receptor A (NPRA), is expressed in cancer cells, and natriuretic peptides have been implicated in cancers. However, the direct role of NPRA signaling in prostate cancer remains unclear.
NPRA expression was examined by western blotting, RT-PCR and immunohistochemistry. NPRA was downregulated by transfection of siRNA, shRNA and NPRA inhibitor (iNPRA). Antitumor efficacy of iNPRA was tested in mice using a TRAMP-C1 xenograft. Here, we demonstrated that NPRA is abundantly expressed on tumorigenic mouse and human prostate cells, but not in nontumorigenic prostate epithelial cells. NPRA expression showed positive correlation with clinical staging in a human PCa tissue microarray. Down-regulation of NPRA by siNPRA or iNPRA induced apoptosis in PCa cells. The mechanism of iNPRA-induced anti-PCa effects was linked to NPRA-induced expression of macrophage migration inhibitory factor (MIF), a proinflammatory cytokine over-expressed in PCa and significantly reduced by siNPRA. Prostate tumor cells implanted in mice deficient in atrial natriuretic peptide receptor A (NPRA-KO) failed to grow, and treatment of TRAMP-C1 xenografts with iNPRA reduced tumor burden and MIF expression. Using the TRAMP spontaneous PCa model, we found that NPRA expression correlated with MIF expression during PCa progression.
Collectively, these results suggest that NPRA promotes PCa development in part by regulating MIF. Our findings also suggest that NPRA is a potential prognostic marker and a target for PCa therapy.

Full-text

Available from: Katherine L Meyer-Siegler
Natriuretic Peptide Receptor A as a Novel Target
for Prostate Cancer
Wang et al.
Wang et al. Molecular Cancer 2011, 10:56
http://www.molecular-cancer.com/content/10/1/56 (17 May 2011)
Page 1
RESEARC H Open Access
Natriuretic Peptide Receptor A as a Novel Target
for Prostate Cancer
Xiaoqin Wang
1
, Payal Raulji
1
, Shyam S Mohapatra
2,6
, Ronil Patel
1
, Gary Hellermann
2
, Xiaoyuan Kong
2
,
Pedro L Vera
3,4
, Katherine L Meyer-Siegler
4
, Domenico Coppola
5
and Subhra Mohapatra
1,6*
Abstract
Background: The receptor for the cardiac hormone atrial natriuretic peptide (ANP), natriuretic peptide receptor A
(NPRA), is expressed in cancer cells, and natriuretic peptides have been implicated in cancers. However, the direct
role of NPRA signaling in prostate cancer remains unclear.
Results: NPRA expression was examined by western blotting, RT-PCR and immunohistochemistry. NPRA was
downregulated by transfection of siRNA, shRNA and NPRA inhibitor (iNPRA). Antitumor efficacy of iNPRA was
tested in mice using a TRAMP-C1 xenograft. Here, we demonstrated that NPRA is abundantly expressed on
tumorigenic mouse and human prostate cells, but not in nontumorigenic prostate epithelial cells. NPRA expression
showed positive correlation with clinical staging in a human PCa tissue microarray. Down-regulation of NPRA by
siNPRA or iNPRA induced apoptosis in PCa cells. The mechanism of iNPRA-induced anti-PCa effects was linked to
NPRA-induced expression of macrophage migration inhibitory factor (MI F), a proinflammatory cytokine over-
expressed in PCa and signi ficantly reduced by siNPRA. Prostate tumor cells implante d in mice deficient in atrial
natriuretic peptide receptor A (NPRA-KO) failed to grow, and treatment of TRAMP-C1 xenografts with iNPRA
reduced tumor burden and MIF expression. Using the TRAMP spontaneous PCa model, we found that NPRA
expression correlated with MIF expression during PCa progression.
Conclusions: Collectively, these results suggest that NPRA promotes PCa development in part by regulating MIF.
Our findings also suggest that NPRA is a potential prognostic marker and a target for PCa therapy.
Introduction
Prostate cancer (PCa) is the third leading cause of death
among men in Amer ica [1,2]. The mortality fro m PCa
results from m etastases to bones and lymph nodes and
progression from androgen-dependent to androgen-
independent disease. While androgen d eprivation ha s
bee n effective in treating androgen-dependent PCa, it is
ineffective in treating advanced P Cas, the primary cause
of mortality. Epidemiological and histopathological stu-
dies have implicated inflammation in the pathogenesis
of PCa [3-5]. Studies have con sistently shown a
decreased risk of PCa among men who regularly take
aspirin or other nonsteroidal anti-inflammatory drugs
(NSAIDs) [6-8]. Despite beneficial effects, the side
effects from using high doses of COX-2 inhibitors for
cancer prevention are a major concern. These observa-
tions emphasize the need for development of new effec-
tive treatments for advanced PCa.
The family of natriuretic peptide hormones has broad
physiologic effects. In additio n to vasodilation, cardio-
vascular homeostasis, sodium excretion and inhibition
of aldosterone secretion, they have been implicated in
immunity and inflammation [9-18]. The effects of atrial
natriuretic peptide (ANP) are mediated by its interaction
with the cell surface natriuretic peptide receptor A
(NPRA; high affinit y) and natriuretic peptide receptor C
(NPRC; low affinity). In patients with prostate tumors,
the immune response plays a large part in the progres-
sion of the disease and it i s likely that the NPRA system
is involved; but the role of NPRA in human cancers
remains unknown. A novel peptide, NP
73-102
, h as been
identified [14] whose sequence is immediately N-term-
inal to the ANP peptide and which is an inhibitor of
NPRA (iNPRA). NP
73-102
does not bind to NPRA but
* Correspondence: smohapa2@health.usf.edu
1
Department of Molecular Medicine, University of South Florida, Tampa FL,
33612, USA
Full list of author information is available at the end of the article
Wang et al. Molecular Cancer 2011, 10:56
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© 2011 Wang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/license s/by/2.0), which permits unres trict ed use, distribution, and reproduction in
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Page 2
blocks its expression, and we have sho wn that it pos-
sesses bronchodilatory, anti-inflammatory [14,16,19,20]
and antitumor activity [19].
We previously reported that mice deficient in NPRA
(NPRA-knockout, KO) exhibit significantly decreased
inflammation [1 6,19-21]. Furthermore, we found that
NPRA-KO mice d o not permit growth of implanted
human lung cancer, melanoma and ovarian cancer cells
[19], suggesting that NPRA may be a novel therapeutic
candidate. Given the ev olutionary conservation of ANP
in many species, we reasoned that NPRA expression
may be relevant in human c ancers. In this study, we
examined the expre ssion of NPRA in PCa cell lines and
human tissue samples and determined whether NPRA
can be used as a target for PCa therapy. The results
show that increased NPRA expression is strongly asso-
ciated with progression of human PCa and that NPRA
deficiency prevents growth of transplanted PCa cells and
inhibits tumor burden in part by downregulating macro-
phage migration inhibitory factor (MIF) in PCa cells.
Results
PCa cells have increased NPRA levels
NPRA expression studies in human tissues have been
limited by lack of availability of appropriate antibodies
to NPRA. The antibodies that are commercially available
are very poor in quality a nd do not provide consistent
results. We developed an antibody to NPRA in rabbits
using a specific antigenic peptide (amino acids 1010-
1031 o f mouse NPRA protein, which is homologo us to
rat and human NPRA). As shown in Figure 1A, an
approximately 130 kDa band corresponding to NPRA
was detected only in human PCa cell lines, PC3 and
DU145 that express NPRA, but not in the RGM1 cell
line that does not express NPRA [22]. The specificity of
the anti-NPRA antibody was confirmed by ELISA (Addi-
tional file 1, Fig. S1A), western blotting (Figure 1A, lane 5)
and by immunofluorescence (Additional file 1, Fig. S1B)
and immunohistochemistry (Additional file 1 Fig.S1C-D).
We examined NPRA expression by western blotting in
various types of PCa tumors and compared it with that
in normal prostate epithelial cells (PrEC and RWPE)
and benign prostatic hyperplasia (BPH) cells. Results of
the western blot show that NPRA is expressed abun-
dan tly in the androgen-dependent PCa cell li ne, LNC aP
and androgen-independent cell lines C4-2, PC3 and
DU145, but not in PrEC cells and only weakly in RWPE
and BPH cells (Figure 1B). Very little NPRA is detected
in the stromal cell line, WPMY, which is derived from
normal prostate. NPRA protein expression in DU145
cells correlated with mRNA level, as verified by real-
time PCR (Figure 1C). Lysates of normal RGM1 cells
that do not express NPRA were used as control. NPRA
is a lso highly expressed in transplantable syngeneic
tumor lines derived from TRAMP (transgenic adenocar-
cinoma mouse prostate) mice which get spontaneous
PCa. NPRA is strongly expressed i n the tumorigeni c
TRAMP-C1 a nd -C2 PCa c ell lines but less abundantly
in the non-tumorigenic TRAMP-C3 PCa cell l ine
(Fig ure 1D) [23]; the latter s hows a three-fold reduction
in growth and colonization potential compared to
TRAMP-C1 and C2 cells (Additio nal file 2, Fig. S2). In
addition, increased N PRA expression was see n in pros-
tate epith elial lines from intac t conditional homozygous
Pten knockout mice (PTEN-CaP2) that are tumorigenic
compared to heterozygous Pten knockout mice (PTEN-
P2) (Figure 1D) [24]. These results suggest that NPRA is
more abundantly expressed in PCa cells than normal or
benign prostate epithelial cells. Expression of the natural
ligand for NPRA, ANP was examined in culture d PCa
cells. ANP expression was detected in culture supern a-
tants of PC3 and DU145 PCa cells and WPMY stromal
cells (Figure 1E) but not in normal prostate epithelial
cells or LNCaP cells. These results suggest that NPRA is
predominantly expressed in prostate tumor cells, while
ANP is expressed in stromal cells and in androgen-inde-
pendent PCa cells, but not in androgen-dependent cells.
NPRA protein expression correlates with human PCa
progression
The clinical relevance of NPRA expression during human
PCa development was examined in BPH, high grade PIN
(prostatic intraepithelial neoplasm) and prostatic adeno-
carcinoma using a human PCa tissue microarray (TMA)
containing 240 samples. The TMA samples included BPH
(n = 24), low grade prostatic intraepithelial neoplasm
(PIN-L) (n = 21), high PIN (PIN-H) (n = 14), prostate car-
cinoma (PC) with a Gleason score of 6 (n = 33), PC with a
Gleason score of 7 (n = 82), PC with a Gleason score of
8 to 10 (n = 51) and androgen-independent (AI) PC (n =
15). The TMA slide was immunostained with a rabbit
anti-human N PRA antibo dy using a Ventana Discovery
XT automated system (Ventana Medical Systems, Tuc-
son, AZ) and the data were statistically analyzed. A
representative image (200×) of one sample from each dis-
ease stage i s shown in Figure 2A. The results demon-
strate that the majority of epithelial cells in BPH and
PIN-Lwereweaklystainedfor NPRA, preferentially in
the nucleus (Figure 2B) and that the PIN-H samples were
weakly to moderat ely p ositive for NPRA. Gleason-6 PCa
samples exhibited moderate to strong NPRA immunoreac-
tivity. Weak and fo cal staining of stromal/inflammatory
cells was also observed in these samples. In contrast,
NPRA staining wa s uniformly strong and prominent and
predominantly localized to the cyto plasm o f the tumor
cells in Gleason 7-10 and in AI PCa samples (Figure 2B).
Stromal/inflammatory cells in these samples also showed
moderate NPRA expression.
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The TMA slide was scored for intensity and cellularity
by an expert pathologist. The final score was classified
as: 0, negative; 1-3, weak; 4-6, moderate; and 7-9, strong.
Figure 2C shows the distribution of scores in each dis-
ease stage. The results show that the mean sample score
increased during PCa progression. The Additional file 3,
Table S1 displays a median analysis of NPRA expression
in the TMA for 240 subjects. Across all 240 subjects,
the median score was 4. Additional file 4, Table S2
shows the frequency in eac h disease group of having a
score falling at or below the median and having one
above the median. The number of observations in the
BPH group with a score >4 was zero, while for Gleason
6, Gleason 7, Gleason 8-10 and AI groups the numbers
were respectively 14 (of 33), 43 (of 82), 34 (of 51) and 8
(of 15). A chi-squared (two-way frequency table) value
of 50.761 with asymptomatic s ignificance of p <0.0001
was obtained, suggesting that the r elationship between
NPRA expression and PCa stage is very strong. A Krus-
kal-Wallis test indicated that the difference in NPRA
expression among t he seven diagnostic groups was
highly significant (p < 0.0001). The pairwise Wilcoxon-
Mann-Whitney tests show that NPRA expression is
strongly associated with PCa progression. The elevated
NPRA expression in high-grade tumors may reflect its
role in tumor-stromal interaction. Since the outcomes of
the Kruskal-Wallis and Wilcoxon-Mann-Whitney tests
are of ordinal value and do not follow the normal distri-
bution that the ANOVA or t-test requires, a nonpara-
metric version of these two methods was used.
NPRA deficiency impairs engraftment of PCa cells
Since, NPRA signaling is involved in inflammation and
the local inflammatory milieu plays a role in PCa devel-
opment, we reasoned that NPRA might be i mportant
for prostate tumor growth . The role of NPRA in modu-
lating PCa progression wastestedusingTRAMP-C1
cells, which form tumors when grafted subcutaneously
into syngeneic C57BL/6 hosts [23]. For in vivo assa ys,
C57BL/6 (WT), NPRA-heterozygous (NPRA-het) and
NPRA-KO mice were injected subcutaneously with
TRAMP-C1 cells. Mice were euthanized seven weeks
100
-actin
kDa
NPRA
140
PC3
DU14
5
RGM1
PC3
PC3
1 2 3 4 5
42
NPRA
-
actin
WPMY
PrEC
RWPE
DU145
PC3
BPH
RGM1
C4-2
LnCaP
PC3
42
kDa
130
C1 C2 C3 P2 CaP2
NPRA
-actin
42
kDa
130
ANP (pg/ml)
0
10
15
20
25
30
5
0
1
2
3
4
5
6
7
8
9
Relative NPR1 expression
A
E
D
B
C
Figure 1 NPRA expression in tumor cells. (A) Immunobl ot analysis demonstrating specifi city of ant i-NPRA antibody. Cell lysates we re
subjected to SDS-PAGE/immunoblot analysis with rabbit polyclonal antibody specific for human NPRA (top) and beta-actin (bottom). For the
competition assay, lane 5 was incubated with NPRA-antibody adsorbed with NPRA peptide (20 μg/ml). (B) Western blotting for NPRA expression.
b-actin expression was used as loading control. (C) Total RNA of indicated cell lines was analyzed for NPRA by real time PCR. NPRA expression
was normalized with respect to b-actin. (D) Whole cell lysates of TRAMP-C1, C2, C3, CaP2 and P2 cells were analyzed by western blotting for
NPRA. (E) ANP expression in PCa cells. Cells were cultured in media containing 0.5% fetal bovine serum for 48 hrs and the culture supernatants
were analyzed for ANP by enzyme immunoassay kit (Phoenix Pharmaceuticals).
Wang et al. Molecular Cancer 2011, 10:56
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after injection and tumor sizes and weights were com-
pared (Figure 3A). TRAMP-C1 cells failed to engraft in
NPRA-KO mice and no visible tumors were detected in
the homozygous group ten weeks after tumor cell injec-
tion. Some t umor growth was observed in NPRA-het
mice, but at a significantly reduc ed level compared to
that in WT C57 BL/6 mice, suggesting that host NPRA
gene dosage is a dete rmining factor for the growth of
tumor cells in these mice. The rol e of NPRA deficiency
in the survival of TRAMP-C1 cells was tested in vitro
by ectopic expre ssion of a plasmid encoding small inter-
feri ng RNA against N PRA (siNPRA). Expression of siN-
PRA-2, but not siNPRA-1, significantly reduced
expression of NPRA (Figure 3B). Apoptosis was detected
by western blotting for PARP cleavage (Figure 3B) and
by the t erminal transferase dUTP ni ck end labeling
(TUNEL) assay (Figure 3C). Downregulation of NPRA
expression by siNPRA-2 induced significant apoptosis in
PCa cells.
NPRA downregulation inhibits MIF expression
We reported previously that NPRA-deficient mice fail to
mount an inflammatory response, as exemplified by the
lack of goblet cell hyperplasia and infiltrat ion of eosino-
phils in the lungs of NPRA-KO mice compared to those
of WT mice, when sensitized and challenged with oval-
bumin [19]. The lack of inflammatory response corre-
lated with reduced levels of inflammatory cytokines IL-
A
B
C
BPH PIN-H Gleason 6
Gleason 7 Gleason 8 Androgen Ind
BPH
Gleason 7
Figure 2 Strong immunoreactivity for NPRA in human prostate TMA. (A) A 200× image of a representative sample from each disease stage
is shown. (B) Images (400×) of a representative BPH and Gleason 7 are shown. (C) The final NPRA scores for each sample of different stages of
PCa are shown. The bar represents the mean sample score for each category of PCa.
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4, IL-5 and IL-6 in the bronchoalveolar lavage (BAL)
fluid of the NPRA-KO mic e relative to that of WT mice
[19,25]. To examine whether the antitumor effects of
iNPRA w ere due to lack of local inflammation in pros-
tate tissue, we injected mice with lipopolysaccharide
(LPS), a potent inducer of local inflammation and com-
pared prostate tissues for alterations in gene expression
in WT and NPRA-KO mice. P rostate tissue was col-
lected from LPS-treated and control mice, and total
RNA was examined for differential gene expression
using a mouse autoimmune and inflammatory response
Oligo GEarray (SuperArray, MD). Analysis of genes
altered more than two-fold during LPS challenge in WT
and NPRA-KO mice identified 24 genes that are either
upregulated (15) or downregulated (9) in the prostate
tissue of NPRA-KO mice compared to their expression
levels in control mice. A few of the genes that are
down-regulated during LPS stimulation in NPRA-KO
mice is shown in Figure 4A, and i nclude: fibronectin 1
(Fn1), which is involved in t he acute phase response
[26], granulin [27] and S100 calcium binding protein A
11 (S100a11) [28], which are cytok ines, IL6 signal trans-
ducer (IL6st; also known as gp130), a cytokine receptor
[29,30] and MIF, which is involved in the inflammatory
response [31].
Since, MIF has been reported to be involved in PCa
progression [31-33], the possibility th at N PRA depletion
modulates MIF expression was tested using shRNAs for
NPRA in TRAMP-C1 cells. As shown in Figure 4B,
transfection of T RAMP-C1 cells with shNPRA-1 and
shNPRA-2 reduced NPRA expression >80% and also
decreased MIF expression >90%. Since overexpression of
plasmid-encoded NP
73-102
downregulates NPRA (Addi-
tional file 5, Fig. S3), pNP
73-102
was also used as an inhi-
bitor of NPRA (iNPRA) in this study. Ectopic expression
of the plasmid encoding NP
73-102
,butnotthepVAX
vector, reduced both NPRA (~40%) and MIF expression
(~50%) in PC3 cells (Figure4C-D)andinTRAMP-C1
cells (data not shown).
iNPRA reduces tumor burden in part by downregulating
MIF
To rule out the possibility that impaired engraftment of
TRAMP-C1 cells in NPRA-KO mice i s due to immune
rejection, we examined the potential of NPRA inhibition
to block the growth of TRAMP-C1 cells in immuno-
competent C57BL/6 mice. Mice were inoculated with
TRAMP-C1 cells and divided into four groups. Two
weeks later, mice in each group were injected i.p. twice
a week with chitosan nanoparticles (CNPs) encapsulat-
ing plasmid DNA (25 μg/mouse) encoding em pty vector
(pVAX), pNP
73-102
, or a con trol peptide encoding
human vessel d ilator (pVD) or a combination of 12.5
μg each of pNP
73-102
and pVD, using methods as
described [19,20]. Mice were monitored for tumor
growth and tumor sizes were recorded on the indi-
cated days (Figure 5A). Tumor growth was significantly
inhibited in mice treated with pNP7
3-102
compared to
pVAX- or pVD-treated groups. Mice were euthanized
on day 65 after treatment, and tumor weights were
measured and compared (Figure 5B). As shown in
Figure 5A-B, a significant reduction (p < 0.05) in
tumor burden was seen in mice treated with 25 μgof
pNP
73-102
but not with the pVAX or pVD plasmids.
Mice treated with 12.5 μgpNP
73-102
showed only mod-
erate inhibition of tumor burden. The plasmid pVD
encodes a peptide corresponding to human VD a nd is
not homologous with mouse VD; thus, lack of any
antitumor effects in pVD-treated mice suggests the
specificity of these peptides in vivo. To understand the
C
A
B
WT Het KO
Tumor Weight (gm)
< 0.05
< 0.01
0
0.1
0.2
0.3
0.4
Clvd. PARP
-actin
NPRA
pU6
Psi-1
Psi-2
42
kDa
130
89
p
U6
p
si-2
TUNEL
DAPI
Figure 3 NPRA-deficiency impairs tumor engraftment and induces apoptosis of PCa cells . ( A) NPRA-deficiency impaired engraftment of
TRAMP-C1 cells. Three groups of mice (wild type (WT), heterozygous (Het) and homozygous (NPRA-KO), (n = 5 per group) were injected s. c. in
the left and right flanks with 5 × 10
6
TRAMP-C1 cells per site. Mice were euthanized ten weeks after injection. Tumors were excised and
weighed. Mean tumor weights ± SEM are shown. (B-C) NPRA deficiency induced apoptosis of PCa cells. TRAMP-C1 cells were transiently
transfected with psiNPRA (si1 and si2) and control plasmid (pU6). Cells were harvested 72 hrs later and whole cell lysates were analysed for
NPRA and PARP by western blotting. (C) TRAMP-C1 cells were transfected with pU6 or psi2 plasmids. Forty-eight hours after transfection,
apoptosis was monitored by TUNEL assay.
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Page 6
B
A
C
D
0
1
2
3
4
5
6
Fn1
Granulin
IL6st
Mif
S100a11
WT
NPRA-KO
0
1
2
3
4
5
6
Fn1
Granulin
IL6st
Mif
S100a11
WT
NPRA-KO
Relative gene expression
C 1 2 NPRA-ps
h
NPRA
MIF
-actin
42
kDa
130
12
kDa
NPRA
MIF
-actin
pVAX phNP
42
130
12
0
0.5
1
1.5
2
pVAX phNP
Relative Intensity
NPRA MIF
Figure 4 NPRA depletion inhibits MIF expression. (A) SuperArray analysis of prostate tissues of NPRA-KO and WT C57BL/6 mice. The relative
expression level of genes that are altered in the prostate tissues of NPRA-KO vs. WT is shown. (B-C) Cells were transfected with iNPRAs. Whole
cell lysates were extracted 72 hrs after transfection and examined for NPRA and MIF by western blotting. TRAMP-C1 cells were transfected with
pshNPRAs or empty vector (B); PC3 cells were transfected with phNP
73-102
plasmid or pVAX plasmid (C). (D) Relative band intensity for NPRA and
MIF expression in Fig. 4C is shown.
C
A
B
Tumor Weight (gm)
**
pVAX pNP pVD pNP +
VD
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
Tumor size (mm
3
)
0
10
20
30
40
50
60
70
80
90
pVAX
pNP73-102
pVD
pNP73-102+pVD
1 34 41 48 55 62
pVAX
pNP
NPRA
MIF
1 2 3 4 5 6
-acti
n
42
kDa
130
12
Figure 5 iNPRA treatment reduces tumor burden by inhibiting MIF expression. (A-B) Effects of iNPRA in TRAMP-C1 inoculated xeno grafts
in immunocompetent mice. Four groups of C57BL/6 mice (n = 7 per group) were injected s.c. in the right flank with 5 × 10
6
TRAMP-C1 cells.
Two weeks later, tumor-inoculated mice were treated with CNPs encapsulated with pVAX, pNP
73-102
, pVD or a combination of pNP
73-102
and
pVD i.p. twice a week until euthanized. The tumor size (A) was measured at the indicated days, and the weight was recorded after tumor
resection (B). (C) NPRA expression correlates with MIF expression in tumor lysates. Tumor lysates from B were analysed for NPRA and MIF by
western blotting. pVAX (lanes 1-3) and pNP
73-102
(lanes 4-6). b-actin was used as a loading control.
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antitumor effects of pNP
73-102
,weexaminedNPRA
and MIF expression in TRAMP-C1-engrafted tumor
lysates from representative control (pVAX) and pNP
73-102
-
treated mice. The results (Figure 5C) show that treatment
of mice with pNP
73-102
, but not with pVAX, significantly
reduced expression of NPRA and MIF; therefore, expres-
sion of these proteins may be linked to growth of primary
tumors in TRAMP-C1-inoculated C57BL/6 mice.
Lastly, we examined NPRA and MIF expression in pri-
mary prostate tumors from TRAMP mice. Western
blots showed that NPRA and MIF are detected in the
lysates of primary prostate tumors from TRAMP mice
of varying ages (18-30 weeks of age) (Figure 6A; lanes 1-
4) but not in prostates from age-matched WT C57BL/6
mice (18 and 28 weeks of age) (Figure 6A; lanes 5-6).
These results suggest that tumor cell lines, as well as
primary prostate tumors of TRAMP mice, show signifi-
cantly higher levels of NPRA and MIF compared to nor-
mal cells or prostate cells from C57BL/6 mice. We also
compared NPRA and MIF expression in total cell lysates
of human PCa cells by western blotting. Results pre-
sented in Figure 6B suggest that increased MIF was seen
in the lysates of PC3 and DU145 cells that express
NPRA abundantly (Figure 1B) compared to the lysates
ofBPHandRWPE.MIFproteinexpressioninPC3and
DU145 cells parallelled with mRNA expression, as
shown by real-time PCR data (Figure 6C). The results of
these s tudies suggest that NPRA regulates MIF expres-
sion in PCa cells.
Discussion
There remain several ov erarching challenges in PCa
research: the lack of specific clinical markers for early
diagnosis and prognosis of PCa and th e need t o identify
drugs that target androgen-indepen dent PCa tumor cells
directly without damaging health y cells. In this study we
show that NPRA is a potential biomarker for PCa and
candidate for PCa therapy.
One important finding of our study is the d emonstra-
tion that NPRA i s significantly over-expressed in mouse
and human PCa ce lls compared to normal cells. Screen-
ing of a hum an PCa tissue microarray containing 240
tissue samples shows that NPRA is also over-expressed
in human tissues including high grade PIN (prostatic
intraepithelial neoplasm) and prostatic adenocarcinoma.
The benign hyperpl astic glands e xhibited significantly
lower NPRA expression than localized PCa s. These data
are consistent with our previous report and with the
data in this study, showing that NPRA is highly
expressed in both human and mouse PCa cell lines and
in advanced PCa tissues, but not in a normal prostate
epithelial cell line or in a benign prostate hyperplasia
epithelial cell line [19,34,35]. It is to be noted that
NPRA was expressed in the androgen-dependent cell
line L NCaP but no t in the stromal cell line, WPMY.
However, expression of ANP was detected in culture
supernatants of PC3 and DU145 PCa cells and WPMY
stromal cells but not in supernatants from normal pros-
tate epithelial cells or LNCaP cells. These results suggest
that ANP produced by stromal cells can signal via
NPRA on androgen-dependent cells (paracrine), whereas
androgen-independent cells produce both ANP and
NPRA and can signal in an autocrine manner. Thus,
ANP-NPRA signaling may play a key role in engaging
PCa cells with stroma during PCa pathogenesis. Hence,
PCa may be bett er managed by inhib iting ANP-NPR A
signaling.
Further, we found a significant association between
NPRA expression and Gleason score and pathological
stage. Results from the tissue array studies show that
NPRA is an in dependent predictor of advanced PCa,
and may therefore be useful as a clinical marker.
A
BC
NPRA
MIF
1 2 3 4 5
TRAMP WT
-actin
kDa
130
42
12
MIF
RWPE
DU145
PC3
BPH
kDa
42
12
-actin
0
1
2
3
4
Relative MIF Expression
Figure 6 NPRA and MIF expression in primary prostate t umors and PCa cel l lines. (A) NPRA and MIF expres sion in primary prostate
tumors. Prostate tissues were homogenized using a polytron and cell lysates were analyzed for NPRA and MIF by western blotting. Lanes 1-3:
lysates of TRAMP prostates. Lanes 4-5: lysates of C57BL/6 prostates. (B) Whole cell lysates of tumor cell lines and control (normal) cells were
analyzed by western blotting. (C) Total RNA of indicated cell lines was analyzed for MIF by real-time PCR.
Wang et al. Molecular Cancer 2011, 10:56
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Although, a number of marker antigens have been
reported for PCa, none of them is specific enough to
pass the clinical test for use in PCa prognosis. Given the
strong positive correlat ion (r = 0.64, p < 0.005) between
NPRA expression and the severity of the clinical stage,
particularly in androgen-independent PCa, NPRA may
prove to be an effective clinical prognostic marker.
Our study also suggests that NPRA may be a drug tar-
get for treating PCa. Using the TRAMP-C1 spontaneous
PCa model, we demonstrated that NPRA-KO mice,
which have normal heart, kidney and vascular function,
have no detectable increase in postnatal mortality, do
not permit growth of implanted PCa cells and have a
normal lifespan of over 24 months. Tumor growth is
observed in NPRA-het mice but at a significantly
reduced level compared to that in WT C57BL/6 mice,
which indicates that host N PRA gen e dosage is a deter-
mining factor for the growth of tumor cells in mice.
This finding is consistent with the reports that atrial
natriuretic f actor peptides (ANP and VD) inhibit the
proliferation of PCa cell s in vitro and in mice [34]. This
is presumably due to the feedback inhibition of NPRA
expression caused by high doses of A NP or other
natriuretic peptides, such as NP
73-102
(our data)
[18,34,36,37]. Thus, while low doses of these peptides
stimulate NPRA signaling, high doses inhibit NP RA sig-
naling and show anticancer effects. In sum, NPRA pro-
vides a heretofore undescribedtargetforPCa.This
hypothesis is also supported by the observation that
NPRA is an upstream regulator of IL-6, which has been
reported as a target for PCa therapy [38,39].
The f inding that pNP
73-102
inhibits NPRA expression
prompted us to examine its role in treating PCa.
TRAMP-C1 cells injected into C57BL/6 mice induced
tumors in the c ontrol mice but not in pNP
73-102
-treated
mice. These findings demonstrate the potential utility of
pNP
73-102
for the treatment of PCa. Although the
mechanism of tumor inhibition by pNP
73-102
is
unknown, the evidence that pNP
73-102
significantly
decreases the expression of NPRA suggests that this
may be the explanation for its antitumor effect. A per-
ceived l imitation in iNPRA therapy for PCa is t he nor-
mal physiological role of NPRA in blood pressure
regulation. To ad dress this issue we compared blood
pressure of NPRA-KO mice with that of TRAMP mice
and found no relationship between NPRA expression,
blood pressure levels and PCa incidence (Additional file
6, Fig. S4), which is consistent with studies in humans
that showed no relationship between blood pressure and
PCa [40,41].
Another major finding of our report is that the antitu-
mor effects of limiting NPRA expression may be due to
a reduction in inflammatio n in the tumor environment.
Our evidence shows that a number of molecules may be
regulated by NPRA signaling including MIF and IL-6,
both of which have been implicated in PCa develop-
ment. Increase d MIF mRNA expression and serum MIF
levels have be en associated with progression of PCa
when tumor and benign tissue from matched samples
were compared [31,33,42]. Elevated IL-6 levels are found
in patients with metastatic PCa and are associated with
a poor prognosis [43]. Furthermore, aberrant expression
of the IL-6 gene and increased production of IL-6 are
associated with advanced bon e metastasis and increased
morbidit y [43-46], as well as resis tance to chemot herapy
[47]. There are three lines of evidence supporting the
idea that NPRA is an upstream regulator of MIF in PCa
cells: ( i) a 2.5-fold reduction in MIF mRNA was found
after LPS treatment of NPRA-KO mice co mpared to
WT mice. (ii) MIF expression was detectable in the
prostate tissues of TRAMP mice, but not in WT mice,
and (iii) N PRA downregulation reduced MIF expression
in cultured TRAMP-C1 cells and xenografts. Consistent
withtheseobservations,aPCatissuearraystainedfor
NPRA showed expression of MIF (data not shown).
Since intratumoral expression of MIF was correlated
with serum IL-6 in patients with non-small cell lung
cancer [48] and IL-6 was shown to be one of the poten-
tial MIF-regulated gen es i n DU145 cells [32], we specu-
late that NPRA signaling may regulate IL-6 in PCa cells
via MIF. In support of this hypothesis, we found ele-
vatedIL-6intheserumofTRAMPmiceduringPCa
development (unpublished observation). These data sup-
port our previously reported studies, where lung tissues
of NPRA-KO mice failed to induce IL-6 during OVA-
induced inflammatory challenge and showed reduced
expression of activated p65- a nd p50-NF-kB [19,20].
Together, these studie s show that NPRA may affect PCa
progression by regulating in part MIF and IL-6 expres-
sion, both of which have been linked to PCa.
In summary, we demonstrate that increased NPRA
expression is strongly associated with progression of
human PCa and that NPRA d eficiency prevents growth
of tr ansplanted PCa cells and inhibits tumor burden in
TRAMP mice in part by downregulating MIF in PCa
cells.
Materials and methods
Materials
Normal prostate epithelial cell line (PrEC) was pur-
chased from Lonza (Allendale). RWPE, WPMY , Tramp-
C1 and PC3 c ells were p urchased from the American
Type Cu lture Collection (Manassas, VA, USA). DU145,
PC3, benign prostatic hyperplasia (BPH), LNCaP, C4-2,
the rat gastric muc osa cell line (RGM1), P2 and CaP2
were described before [22,24,32,49]. The beta-actin anti-
body was obtained from Sigma, the PARP antibody
from Santa Cruz Biotechnology, and the MIF antibody
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from Abcam. Lipofectamine 2000 reagent was obtained
from Invitrogen.
NPRA antibody production and purification
Antibody against NPRA was generated by injecting rab-
bits (New Zealand white) with 400 μg of synthetic
NPRA peptide (amino acid 1010-1031 of mouse NPRA
protein, which is homologous to rat and human NPRA)
conjugated to keyhole limpet hemocyanin (BioSynthesis,
Inc., Lewisville TX). Antibody was purified by applying
serum to a column of protein A/G agarose (Invitrogen,
Carlsbad, CA) equilibrated with 20 mM Tris, pH 7.5,
150 mM NaCl, and eluting with 100 mM citrate, pH
3.0. The eluate was neutralized with 5 M NaOH, gly-
cerol was added to 50% and the purified aliquots were
stored at -20°C.
NPRA antibody competition assay
For determining NPRA antibody titer, a 96 well plate
was coated with the non-KLH-conjugated NPRA-speci-
fic peptide (amino acids 1010-1031 of mouse NPRA
protein) that was used to raise the ant ibody or an unre-
lated peptide. Rabbit sera from 6 animals were pooled
and purified using a protein A/G sepharose column
(Pierce). A serial dilution of the ant ibody was added to
each well of a microtiter plate coated with peptides
overnight. For the competition assay, purified antibody
was incubated with NPRA-specific peptide on ice for 1
hr and then added to the plate. The plate was washed
and developed using HRP-conjugated anti-rabbit IgG
(Cell Signaling) and HRP-substrate (R & D Systems).
The pl ate was read at 450 nm using a Synergy H4 p late
rea der (Biotek). The values presente d are means of four
wells.
Cell counting and colony assay
At the indicated times, cells were harvested by trypsini-
zation and viable cell numbers enumerated by trypan
blue dye-exclusion. To test colonization potential, TR-
C1or TR-C3 cells were plated in 100 mm dishes at 1000
cells/plate. After 3 weeks, the resulting colonies were
stained with 0.25% crystal violet, photographed and
counted.
Luciferase reporter assays
PC3 cells were co-transfected with hNP
73-102
, mNP
73-102
or vector alone (pVAX), reporter plasmid (pNPRA-Luc),
and a transfection normalization vector (pRenilla-luc).
DNA (0.5-1 μg/10
6
cells) was transfected into 60% con-
fluent PC3 cells using lipofectamine (Life Technologies).
Forty-eight hours after transfection, the reporter activity
was measured with the Dual-Luciferase Reporter assay
system (Promega) according to the manufacturer s
instructions. Luminescent signals were quantified with
the Synergy H4 (Biotek). Reporter assay results were
based on data averaged from three replicates.
Tissue microarray (TMA) staining
A human prostate cancer TMA containing 240 samples,
prepared in the histology laboratory of the Moffitt Can-
cerCenterTissueCoreFacilitywasusedtotestfor
expression of NPRA and MIF. The TMA slide was
stained using a Ventana Discovery XT automated sys-
tem (Ventana Medical Systems, Tucso n, AZ) , according
to the manufacturer s protocol. Briefly, slides were
deparaffinized on the automated system with EZ Prep
solution (Ventana). Following heat-induced antigen
retrieval, the slide was incubated with NPRA antibody
(1:300) for 32 min and Ventana anti-rabbit or anti-goat
secondary antibody f or 20 min. The detection system
used was the Ventana OmniMap kit, and the slide was
then counterstained with hematoxylin and dehydrated.
TMA data analysis
The TMA slide was scored for intensity and cellularity by
an expert pathologist. Positive staining for NPRA was
scored into four grades, according to the intensity: 0, 1+, 2
+ and 3+. The percentage of NPRA-positive cells was
score d into three categories: 1 (0-33%), 2 (34-64%) and 3
(65-100%). The product of the intensity and percentage
scores was used as the final score. The final score was clas-
sified as: 0, negative; 1-3, weak; 4-6, moderate; and 7-9,
strong. A median analysis of NPRA scores and the fre-
quency in each disease group of having a score at or below
the median was performed. Also, the chi-squared test, the
Kruskal-Wallis test and the Wilcoxon-Mann-Whitney test
were used to compare the scores by groups. Comparisons
were done for (1) PIN-L vs. BPH; (2) PIN-H vs. BPH; (3)
Gleason-6 vs. BPH; (4) Glea son-7 vs. BPH; (5) Gleason-8
to 10 vs. BPH and (6) AI vs. BPH.
Animals
Male C57BL/6 mice were purchased from the National
Cancer Institute. Male C57BL/6 NPRA-KO or NPRA-
het were described before [19]. All mice were main-
tained in a pathogen-free environment and all proce-
dures were reviewed and approved by the University of
South Florida Institutional Animal Care and Use
Committee.
Preparation of plasmid nanoparticles and administration
to mice
Plasmids encoding NP
73-102
,hNP
73-102
and VD were
constructed as described previously [14,19]. Plasmids
encoding siRNAs against NPRA were described pre-
viously [19]. Plasmids encoding shNPRAs were pur-
chased from Origene. For transfection, epithelial cells at
60% confluence (log phase) were incubated in complete
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medium at 37°C with plasmid DNA (1 μg/10
6
cells)
complexed with lipofectamine (GibcoBRL Life Technol-
ogies, Carlsb ad, CA). For tumor cell inocul ation,
TRAMP-C1 ce lls were t rypsinized, washed and resus-
pended in PBS at 5 × 10
7
cells per ml. Mice were
injected s.c. in the flank with 100 μL of resuspended
cancer cells. For evaluating the effects of i NPRA in
modulating tumor progression, plasmids encapsulated in
chitosa n nanoparticles (25 μg of plasmid plus 125 μgof
chitosan) were administered i.p. twice a week until sacri-
ficed. Tumor sizes were measured externally by calipers,
and at the end of experiment (day 62), t he mice were
euthanized and the tumors were removed and weighed.
Proteins from tumors were extracted and examined for
NPRA and MIF expression by western blotting.
Blood pressure measurement
Diastolic and systolic pressures of age-matched mice
were measured using the CODA noninvasive blood
pressure system (Kent Scientific). Briefly, m ice were
placed in a restrainer o n a hot water blanket and the
restrainer was covered with a warm water glove. The
occlusion c uff was fitted to the base of the tail and the
VPR cuff slid down until it reached the occlusion cuff.
Maximum occlusion pressure was set to 250 μLwitha
def lation time of 20 seconds and a minimum volume of
blood flow in the t ail of 10 μL. The occlusion cuff was
inflated to impede the blood flow to the t ail. As the
occlusion cuff is deflated, a second tail cuff with the
VPR sensors records the pressure at the point where
blood flow returns. The systolic is measured at t he first
appearance of tail swelling and the diastolic is calculated
when the increasing rate of swelling ceases in the tail.
Western blot analysis
Western blot assay was performed as previously
described [49]. Cells were lysed, total cellular protein
(120 μg) was separated by SDS-PAGE, blotted to nitro-
cellulose, and incubated with antibodies to specific pro-
teins. Bands were visualized by enhanced
chemiluminescence (Amersha m Life Sciences, Piscat-
away, NJ) on Kodak X-OMAT-AR film.
Real-time PCR analysis
Total RNA was isolated using the RNeasy mini kit (Qia-
gen). One tube cDNA synthesis followed by real-time PCR
was performed in a 25 μl reaction mixture using Taqman
RNA-to-CT 1-Step Kit (Applied Biosystems). Quantita-
tive re al-time PCR was carrie d out on the CFX96 real-
time System (Bio-Rad). Taqman gene expression assays
(Applied Biosystems) Hs00418568, Hs00236988 and
4333762, respectively are used for amplification of NPR1,
MIF and b-actin. The conditions for the real-time PCR
assay were 15 min at 48°C, 10 min at 95°C, 40 cycles of 15
sec at 95°C and 60 sec at 60°C. Expressio n of each target
mRNA relative to b-actin was calculated under experi-
mental and control conditions based on threshold cycle
(C
t
)as
r
=2
(C
t
)
, where ΔC
t
= C
t target
- C
t b-actin
and Δ
(ΔC
t
)=ΔC
t experimental
- C
tcontrol
.
ANP ELISA
Duplicate aliquots of 50 μl of culture supernatants were
assayed for ANP concentration using a fluorescent immu-
noassay kit (Phoenix Pharmaceuticals, Burlingame CA).
ANP standards were run to generate a standard curve that
was used to calculate the average ANP concentration.
SuperArray analysis of prostate tissues
NPRA-KO and WT C57BL/6 mice (n = 4) were injected
i.p. with LPS (1 mg/kg body weight) for 3 hrs, prior to
prostate harvesting. Total RNA was isolated using an
RNAeasy kit (QIAGEN, Valencia, CA) and a pool of
total RNA by group hybridized to the mouse autoim-
mune and inflammatory response Oligo GEarray (Super-
Array Frederick, MD), according to the manufacturer s
instructions. The X-ray films were scanned, and the
spots were analyzed using SuperArray Software. The
relative expression level was determined by comparing
the s ignal intensity of each gene in the array a fter nor-
malization to the signal of a set of housekeeping genes.
Statistics
The number of mice used in each test group was a
minimum of four. Experiment s were repeated at least
once, a nd measurement s were expressed as means ±
SD. Pairs of groups were compared through the use of
Students t tests. Differences between groups were con-
sidered significant at p 0.05.
Additional material
Additional file 1: Fig. S1. Characterization of rabbit polyclonal
antibody to NPRA. (A) NPRA competition assay. Reactivity of of anti-
NPRA antibody to an unrelated (U) peptide and NPRA-peptide (S) is
shown. For the competition assay, NPRA-antibody was adsorbed with
NPRA peptide (20 ug/ml) (referred to as S-A) prior to incubation. (B-D)
Immunofluorescence (B) and immunohistochemistry (C-D) of anti-NPRA
antibody. The indicated cell lines were cultured on chamber slides and
immunostained using anti-NPRA Ab. As a negative control, PC3 cells
were incubated with secondary Ab alone (Control). (C) Two identical
multi-tissue TMA slides containing colon, prostate, breast, and pancreas
tumor tissues were used to optimize immunostaining. TMAs slides were
incubated with NPRA-Ab (left side) or no antibody (right side). (D)
Demonstrate specificity of NPRA antibody. Identical tumor tissues were
immunostained with either NPRA antibody (top) or NPRA-antibody
adsorbed with NPRA peptide (20 ug/ml).
Additional file 2: Fig. S2. Evaluation of TRAMP tumor cell growth
potential and colony-forming ability. (A) Viability counts of tumor cells
after four days. TRAMP-C1, -C2 and -C3 cells were plated at 10
5
cells per
plate for 4 days and viable cell numbers were enumerated at the
indicated days by trypan blue dye-exclusion. (B & C) Tumor cell colony
formation after three weeks. TRAMP-C1 or TR-C3 cells were plated in 100
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mm dishes at 1000 cells/dish. After 3 weeks, the colonies were stained,
photographed (B) or counted (C).
Additional file 3: Table S1. Median analysis of NPRA expression in
tissue multi-array from 240 subjects.
Additional file 4: Table S2. Frequency of Gleason scores above and
below the median.
Additional file 5: Fig. S3. pNP
73-102
inhibits NPRA expression. PC3
cells were co-transfected with pVAX, phNP73-102, pVD or pmNP73-102
and pNPRA-luc plasmid and pRenilla-luc plasmids. Forty-eight hrs after
transfection, lysates were analyzed for luciferase reporter activity. Relative
luciferase activity ± SD is shown.
Additional file 6: Fig. S4. Blood pressure measurements in NPRA
knockout mice compared to wild type and TRAMP mice. Diastolic
and systolic pressure of age-matched wt (n = 3), NPRA-KO (n = 4) and
TRAMP (n = 4) male mice were measured using the CODA noninvasive
blood pressure system (Kent Scientific). Data is presented as mean
pressure ± SD.
Abbreviations
ANP: atrial natriuretic peptide; CNP: chitosan NP; het: heterozygous; KO:
knockout; MIF: macrophage inhibitory factor; NP: nanoparticles; NPRA:
natriuretic peptide receptor A; PCa: prostate cancer; PIN: prostatic
intraepithelial neoplasia; siRNA: small interfering RNA; TRAMP: transgenic
adenocarcinoma mouse prostate; WT: wild type.
Acknowledgements
This research was supported by funds from NIHCA139785 and 9BW-08
Bankhead Coley Cancer Research Program grants to SM and NIHCA152005
to both SSM and SM. We thank Dr. Hong Wu (UCLA) for providing P2 and
CaP2 cell lines, Dr. Wenlong Bai (USF) for TRAMP-C2 cell line and Dr. William
Gower (JAH-VA) for RGM1 cell line. We thank Dr. Hongyu Zheng and Mr.
Murali Kanakenahalli for technical assistance, Drs. Ren Chen and Ambuj
Kumar for Statistical analyses, Dr. Alex Lopez for TMA analysis. We
acknowledge the assistance of the Moffitt Cancer Center Tissue Core,
Microscopy Core and Molecular Biology Core.
Author details
1
Department of Molecular Medicine, University of South Florida, Tampa FL,
33612, USA.
2
Department of Internal Medicine, University of South Florida,
Tampa FL, 33612, USA.
3
Department of Surgery, University of South Florida,
Tampa FL, 33612, USA.
4
Bay Pines Veterans Affairs Healthcare System, Bay
Pines, FL 33744, USA.
5
H. Lee Moffitt Cancer Center.
6
James A. Haley
Veterans Hospital, Tampa FL, 33612, USA.
Authors contributions
XW designed the experiments, interpreted the results and prepared the
manuscript under the supervision of SM. PR, RP, GH and XK provided
technical assistance for experiments. GH also contributed to the manuscript
editing. DC provided expertise in scoring TMA slides. KLM and PLD provided
expertise in MIF related studies. SSM provided key reagents for the study.
SSM, KLM and PLD critically evaluated the manuscript. All authors read and
approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 21 December 2010 Accepted: 17 May 2011
Published: 17 May 2011
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